The present invention relates to a process for delivery of biological materials into living cells and tissues of plants and animals. More particularly, this invention relates to a process wherein, the biomolecules to be delivered into cells are coated on tiny inert particles, called microprojectiles, which are then bombarded on the target cells or tissues. The microprojectiles are coated with biological materials, like nucleic acids and delivered into cells, using a microprojectile gun, also called as xe2x80x98gene gunxe2x80x99 or xe2x80x98biolistic gunxe2x80x99. Commonly used microprojectiles are gold and tungsten particles. The improved process claimed here gives highly efficient, more uniform and reproducible delivery of microprojectiles. As a result, it gives several fold higher expression of the nucleic acid delivered into cells.
Accelerated metal particles coated with nucleic acids are extensively used for introducing genes into intact plants and animals, tissues, cells and organelles (Sanford et al., 1993 Sanford J. C., Smith F. C. and Russell, J. A. 1993 Optimising the biolistic process for different biological applications. Methods in Enzymology, 217, 485-510). A variety of particle guns that deliver microprojectiles into living cells have been described in earlier inventions (U.S. Pat. Nos. 5,100,792, 5,179,022, 5,204,253 and 5,865,796). Though several different types of particles have been discussed, including high density metals like gold, tungsten, platinum, ferrite etc., and low density synthetic polymeric particles (U.S. Pat. No. 5,783,567), the former group has been more successful apparently because these can be delivered with higher kinetic energy and can therefore, go deeper into the target tissue. Gold particles have been more commonly used because gold is more inert than others, i.e., it does not interfere with biological processes and the gold particles have more uniform surface, desirable for minimum cell damage.
The particles are coated with the biological material to be delivered into living cells. The microprojecticles, coated with the biological material are then propelled at high speed, using one of a variety of particle guns made by certain research groups or available commercially.
Accelerated metal particles penetrate several layers deep into tissue, depending upon the velocity and momentum at which these strike the target tissue. The acceleration to particles can be provided by mechanical impulse, centripetal force, electric discharge (U.S. Pat. No. 5,100,792), firing explosives (U.S. Pat. No. 5,179,022) or compressed gas (U.S. Pat. No. 5,865,796) or any other process. The most commonly used and commercially available apparatus for acceleration of microprojectiles involves acceleration using gas shock (U.S. Pat. Nos. 5,204,253 and 5,865,796) and is available commercially from M/S Bio-Rad Laboratories, Hercules, Calif. 94547, USA.
Examples of biological substances that can be coated onto or impregnated into inert particles include biological stain such as fluorescent or radioactive probes, viruses, organelles, proteins, hormones and nucleic, acids. In certain cases delivery of molecules into cells and tissues under liquid pressure has also been claimed (U.S. Pat. No. 5,766,901).
The effectiveness of the delivery of particles is most conveniently measured by examining the expression of a reporter gene delivered in form of DNA coated on such particles. One of the very commonly used reporter genes is uidA (Jefferson, R. A. and Wilson, K. J. 1991. The GUS gene fusion system In: S. Gelvin and R. Schilperoort Ed. Plant Molecular Biology, Kluwer Academic Publishers, Dordrecht, B 14, pp. 1-33) which codes for an enzyme called glucuronidase. Once the gene is inside a cell and if the cell is viable, the gene makes the enzyme glucuronidase. Following bombardment of uidA gene containing particles, if a plant leaf is treated with a chromogenic substrate 5-bromo-4-chloro-3 indoyl-xcex2-D-glucuronic acid, also called X-gluc, it is converted into a blue product due to activity of glucuronidase. The blue spots appear on the bombarded leaf and can be counted by treating the leaf with X-gluc, one to several days after bombardment. Therefore, the number of spots indicate the number of foci within target tissue where particles get delivered without causing significant damage to biological function of cells. Hence, expression of glucuronidase is indicative of delivery of the microprojectile particles in a manner that did not cause damage to biological function of the cell. Therefore, efficiency of biologically effective delivery of microprojectile particles can be measured by counting under microscope, blue spots on the leaf surface for example, in case of plants (or any other tissue) that are formed due to expression of glucuronidase. This method is called as xe2x80x98histochemical methodxe2x80x99 since it allows seeing the activity by color. It is not quantitative but qualitative since it tells the presence of activity but does not quantitatively measure the level of glucuronidase activity. Details of this method, as applied to gold and tungsten coated particles are also given in Ratnayaka and Oard (1995, I. J. S. Ratnayaka and J. H. Oard 1995 A rapid method to monitor DNA precipitation onto microcarriers before particle bombardment, Plant Cell Reports, 14, 794-798).
A second more sensitive method to measure the activity of xcex2-glucuronidase after microprojectile bombardment is by quantitatively measuring the formation of a fluorescent product. In this method, a non fluorescent substrate called 4-methlyumbelliferyl-xcex2-D-glucuronide, also called MUG, is converted by glucuronidase into a fluorescent product called MU. Hence, the increase in fluorescence per mg protein in leaf gives quantitative expression of gene delivery in a manner that did not damage biological function of the target, cells. The less the expression, the less successful the delivery of DNA coated on microprojectiles. The fluorometeric as well as histochemical GUS assays are also described in Kloti et al (1999, A. Kloti, C. Henrich and others in Plant Molecular Biology, 40, 249-266). Both of these standard methods were employed in this invention to illustrate an amazingly high improvement of results obtained by the bombardment protocol claimed by us.
While coating microprojectiles with DNA the procedure recommended by Bio-Rad Laboratories in their catalogue or its minor variations are commonly used. This method is based on the original method developed by Sanford et al. (1993, J. C. Sanford, F. D. Smith and J. A. Russell, 1993 optimising the biolistic process for different biological applications, Meth. Enzymol. 217, 483-509). In this method, 3 mg gold particles (1 xcexcm diameter particles for example, supplied by, Bio-Rad Laboratories, USA) are placed in a microcentrifuge tube and vortexed for 3 min in 0.5 ml 70% ethanol (v/v). The suspension is held at room temperature for 10 min, centrifuged (15000 rpm) for 5 sec and decanted. The pellet of particles is washed three times with 500 xcexcl sterile distilled water. Between two washings, the suspension is mixed thoroughly by vortexing for 1 min, the particles are allowed to settle on bench for 1 min and then centrifuged for 5 sec. Finally, the washed particles are suspended in 50 xcexcl of 50% glycerol. The suspended particles are then coated with DNA by adding 5 xcexcl DNA (1 xcexcg/xcexcl in water), 50 xcexcl CaCl2 (2.5 M) and 20 xcexcl spermidine (0.1 M stock) in that order, vortexed for 3 min, held at room temperature for 5 min, and finally the coated particles are pelleted by pulse centrifugation. The preparation is then washed with 150 xcexcl of 70% ethanol, followed by absolute ethanol before they are suspended in 48 xcexcl ethanol. The coated particles are then vortexed for 1 to 2 seconds before placing 8 xcexcl aliquots on macrocarrier for bombardment on a target tissue, like tobacco leaf. The above commercially recommended procedure given by Bio-Rad Laboratories Ltd. Along with the Helium Drive PDS-1000/He system Biolistic Gun is stated in US/EG Bulletin 1688 by BioRad Life Science Group, California. Hunold et al. (1994, R. Hunold, R. Bronner and G. Hahne, 1994 Early events in microprojectile bombardment: Cell Viability and Particle location. Plant J. 5, 593-604) suggested minor variation in the above described method developed by Sanford. The method of Hunold et al. (1994) is also used commonly. The method of Hunold et al. (1994) was used in this study and is referred to as xe2x80x9cStandard Methodxe2x80x9d. In the standard method, 3 mg gold particles (1 xcexcm diameter particles for example, supplied by, the Bio-Rad Laboratories, USA) are placed in a microcentrifuge tube and vortexed for 3 min in 0.5 ml 70% ethanol (v/v). The suspension is sonicated for 2 sec in water bath sonicator (Branson, USA), held at room temperature for 15 min, centrifuged (15000 rpm) for 5 sec and decanted. The pellet of particles is washed three times with 500 xcexcl sterile distilled water. Between two washings, the suspension is mixed thoroughly by vortexing for 1 min, the particles are allowed to settle on bench for 1 min and then centrifuged for 5 sec. Finally, the washed particles are suspended in 50 xcexcl of 50% glycerol and sonicated for 2 sec in water bath sonicator (Branson, USA). The suspended particles are then coated with DNA by adding 5 xcexcl DNA (1 xcexcg/xcexcl in water), 50 xcexcl CaCl2 (2.5 M) and 20 xcexcl spermidine (0.1 M stock) in that order, vortexed for 3 min, held at room temperature for 5 min, and finally the coated particles are pelleted by pulse centrifugation. The preparation is then washed with 150 xcexcl of 70% ethanol, followed by absolute ethanol before they are suspended in 48 xcexcl ethanol. The coated particles are then vortexed for 1 to 2 seconds before placing 8 xcexcl aliquots on macrocarrier for bombardment, for example, at 1100 psi on tobacco leaf placed at 12.3 cm target distance. A helium driven particle gun PDS-1000He (Bio-Rad, U.S.A.) is commonly used. In this study, the bombarded leaves were incubated on MS agar medium. The glucuronidase activity was examined histochemically by counting the total number of blue spots as well as measured quantitatively fluorimetrically, as in published procedures cited above. A few other minor variations of the above procedure for coating microprojectiles with DNA are given in several references including their applications to maize cells (Klein et al 1988, BioTechnology, 6, 559-563), tobacco plastids (Svab et al, 1990, Proc. Natl. Acad. Sci USA, 87, 8526-8530), tobacco cells (Russell et al, 1992 In vitro Cell Dev. Biol. 28 P, 97-105), micro-organisms and animals (Klein et al, BioTechnology, 10, 286-291), mammalian cells (Heiser, 1994, Analytical Biochemistry, 217, 185-196), plant pollens (Stoger et al, 1995, Plant Cell Reports, 14, 273-278) and several others. Standardization is required to optimize the pressure at which the particles are bombarded on target tissue, At a given particle acceleration (determined by gas pressure and the distance between the point of discharge of particles and the target tissue), the conditions discussed in the above modified procedures make minor differences in efficiency of biologically active DNA delivery into target tissue.
A major difficulty encountered in the bombardment of microprojectiles in the above studies including the above described methods by Sanford et al. (1993) and Hunold et al. (1994), is that the particles of heavy metals like gold and tungsten agglomerate, leading to their non uniform spread following bombardment on the target tissue. The tendency of particles to stick to one another results in the formation of clumps. The particles also stick to the walls of container, like polypropylene tubes in which these are prepared. Sticking to the container and to one another results in loss of substantial amount of particles and DNA and variability in the precipitation of DNA and non reproducible shot-to-shot results (Vain et al., 1993 Plant Cell, Tissue and Organ Culture, 33, 237-246). Delivery of clumps on tissues also results in irreparable damage to target tissue (Ratnayaka and Oard, 1995 Plant Cell Reports 14, 794-798). Use of certain expensive brands of microcentrifuge tubes has been suggested (Sanford et al., 1993 Methods in Enzymology, 217, 483-510) to reduce sticking of the particles to surface of the tube. Although vigorous vortexing, ultrasonication, use of glycerol or polyethylene glycoll are claimed to reduce aggregation, uniform and efficient delivery of the microprojectiles for reproducible results continues to be a major problem (Sanford et al., 1993 Methods in Enzymology, 217, 483-510). Variability in the results from individual bombardment events is so high that an internal control is used in all experiments to allow normalization of such variations between independent experiments, treatments and replicates (see for example, Bruce et al, 1989, Proc. Natl. Acad. Sci USA, 86: 9692-9696; Schledzewski and Mendel, 1994, Transgenic Research 3, 249-255; Schenk et al, 1998, Plant Mol. Biol. Reporter, 16, 313-322).
In a recent patent (U.S. Pat. No. 5,879,918 dated Mar. 9, 1999) Tomes and coworkers claimed an improvement wherein they xe2x80x98cleanedxe2x80x99 the tungsten beads with strong nitric acid while agitating those continuously by sonication during the pretreatment. The pretreatment claimed by them gave an average of about two to thee fold improvement in the expression of xcex2-glucuronidase, following the delivery of reporter gene DNA using tungsten or gold particles on cells of maize plant. The gain was rather small and the shot to shot variation was not controlled by the method claimed by them. Our invention relates to a new method for pretreatment of the metal particles and coating of DNA. The method described here gives 43 to 63 fold enhancement in delivery of the particles, depending upon the method of measurement and the batch of particles used to deliver DNA molecules onto plant leaf tissue. Our improved method does not involve use of strong acids like nitric acid as a step of pretreatment. It reduces shot-to-shot variation to the statistically acceptance level of average 6.7% as compared with the unacceptable variation of 43% obtained by the standard process.
The main object of the invention is to develop a method for uniform and efficient delivery of metal microprojectiles into living cells by bombarding the cells with such particles. The preferred particles are gold particles.
Another object of the present invention allows coating of the gold particles with biological material, preferred biological material being DNA, in such a manner that the particles do not stick to one another or to the walls of container, being polypropylene tube as the preferred container.
The present invention achieves the above objectives by heating the gold particles in dry oven to high temperature, prior to coating with DNA and by substituting ethanol with isopropanol while coating the particles with DNA. Using Helium driven PDS 1000/He (BioRad Laboratories) as the preferred biolistic gun, the resultant particles are delivered extremely efficiently, uniformly and reproducibly in the cells of leaf tissue, used as the preferred biological tissue. Dry heat at high temperature apparently removes the water molecules and possibly other volatile substances that are tightly adsorbed on gold particles. Removal of such molecules prevents agglomeration of gold particles, thus giving highly uniform spread of the particles in target tissues. The uniform spread also prevents the damage caused to tissue if the particles are bombarded as bigger clumps. The DNA delivered by the new method expresses in the target tissue at levels 20 to 70 fold higher than that by the commonly used and recommended methods and publicly published by BioRad Life Science Group and several others. The improved method works highly efficiently irrespective of the conditions under which the particles were stored prior to the usage.
In the process claimed here, the preferred gold particles were first pretreated by heating those at high temperature in a dry oven for several hours before preparing those for coating with DNA. The temperature of heating and the duration of heating are not critical. High temperature and sufficient time are required that would permit the removal (including the removal of water molecules) of unknown interfering residues that adhere to the gold (or tungsten) particles. It is also possible that the heating makes certain chemical or physico chemical changes on the surface of particles. Such undesired molecules may get adsorbed from the surroundings or may get formed on metal particles during their manufacture, transportation or storage. At the lower end, heating for 1 hour at 150xc2x0 C. was helpful. Routinely, the particles were pretreated by heating at 150xc2x0 C. for over-night. The temperature of heating was not critical but generally can range from 80xc2x0 C. to 200xc2x0 C. or higher.
The preheated particles were then used to coat with DNA by a standard procedure except that ethanol was substituted at all steps with isopropanol. A standard procedure (Sanford et al., 1993, Methods in Enzymology, 217, 483-510) is as follows:
In a xe2x80x9cStandard Methodxe2x80x9d, as described earlier 3 mg gold particles (1 xcexcm diameter particles for example, supplied by, Bio-Rad Laboratories, USA) are placed in a microcentrifuge tube and vortexed for 3 min in 0.5 ml 70% ethanol (v/v). The suspension is sonicated for 2 sec in water bath sonicator (Branson, USA), held at room temperature for 15 min, centrifuged (15000 rpm) for 5 sec and decanted. The pellet of particles is washed three times with 500 xcexcl sterile distilled, water. Between two washings, the suspension is mixed thoroughly by vortexing for 1 min, the particles are allowed to settle on bench for 1 min and then centrifuged for 5 sec. Finally, the washed particles are suspended in 50 xcexcl of 50% glycerol and sonicated for 2 sec in water bath sonicator Branson, USA). The suspended particles are then coated with DNA by adding 5 xcexcl DNA (1 xcexcg/xcexcl in water), 50 xcexcl CaCl2 (2.5 M) and 20 xcexcl spermidine (0.1 M stock) in that order, vortexed for 3 min, held at room temperature for 5 min, and finally the coated particles are pelleted by pulse centrifugation. The preparation is then washed with 150 xcexcl of 70% ethanol, followed by absolute ethanol before they are suspended in 48 xcexcl ethanol. The coated particles are then vortexed for 1 to 2 seconds before placing 8 xcexcl aliquots on macrocarrier for bombardment, for example, at 1100 psi on tobacco leaf placed at 12.3 cm target distance. A helium driven particle gun PDS-1000He (Bio-Rad, U.S.A.) is commonly used. In this example, the bombarded leaves are incubated on MS agar medium. The glucuronidase activity is examined histochemically by counting the total number of blue spots as well as is measured quantitatively fluorimetrically, as in published procedures (Jafferson and Wilson, 1991 In Plant Molecular Biology Manual, Kluwer pp. 1-33). In the improved procedure claimed by us, all other steps were executed as described above, except that the gold particles were heated at 90 to 200xc2x0 C. over-night in a glass tube before use in the above procedure and that ethanol was substituted with isopropanol, HPLC (Spectrochem, India) grade in the steps described above. Other alcohols like isopropanol (primary alcohol) and isoforms of butanol, specially volatile alcohols with low solubility in water and acetone were tried and are obvious alternatives that may work at varying efficiencies.
Accordingly the invention provides, an improved process for transporting a biological material into living cells which comprises bombarding the said cells with a biological material coated on metal bead particles, wherein the improvement comprises in pretreating the metal bead particles by heating the said particles at a temperature ranging between 90-300xc2x0 C. for a period ranging between 1 to 18 hours and thereafter coating the said pretreated bead particles with a biological material using an organic solvent.
In an embodiment of the present invention, the heating of the particles is carried out in a dry oven to allow removal of volatile materials.
In an embodiment of the present invention, the particles used are beads selected from any biologically non reactive materials from the group consisting of gold, palladium, platinum or any alloy thereof.
In an embodiment of the present invention, the particles used are beads selected from any biologically non reactive materials from the group consisting of gold, palladium, platinum or any alloy thereof which may be either new or old.
In another embodiment of the present invention, the old particles used are beads selected from any biologically non reactive materials from the group consisting of gold, palladium, platinum or any alloy thereof stored for a period of more than six months.
In another embodiment of the present invention, the beads used have a diameter of from about 0.1 microns to about 3.0 microns.
In yet another embodiment of the present invention, the heat pretreated particles are used for coating of biological material using standard protocols for coating DNA or RNA or other biologically active molecules on metal particles to be used for delivery into cells.
In yet another embodiment of the present invention, the heat treated particles are used for coating of biological materials using isopropanol to prevent clumping of particles or their sticking to walls of the container.
In still another embodiment of the present invention, the particles with or without heat pretreatment are coated with a biologically material by known methods.
In an advantageous embodiment of the present invention, the biological material used is DNA or RNA or another biologically active molecule to be delivered in cells or tissue using microprojectiles as carriers.
In another advantageous embodiment of the present invention, the biological molecules are delivered in the cell to study transient or long term effects of the delivery of the molecules on the living systems including vaccination and drug delivery and their effect within that or in subsequent generations.